A mouse model for β-thalassemia

A mutation that produces an absolute deficiency of normal β-major globin polypeptides has been recovered from a DBA/2J male mouse. Most mice homozygous for the deficiency survived to adulthood and reproduced but were smaller at birth than their littermates and demonstrated a hypochromic, microcytic anemia with severe anisocytosis, poikilocytosis, and reticulocytosis and the presence of inclusion bodies in a high proportion of circulating erythrocytes. Mice heterozygous for the deficiency demonstrated a mild reticulocytosis but were not clinically anemic. Analysis of globin chain synthesis in vitro by ³H-leucine incorporation revealed that β-globin synthesis was nearly normal (95%) in heterozygotes and about 75% of normal in deficiency homozygotes. Molecular characterization of the mutation by restriction analysis revealed a deletion of about 3.3 kb of DNA, including regulatory sequences and all coding blocks for β-major globin. Based on genetic and hematological criteria, mice homozygous for the mutant allele, designated Hbb^th-1, represent the first animal model of β-thalassemia (Cooley's anemia), a severe genetic disease of humans.     

Synthesis of erythrocyte-specific proteins in cultured friend leukemia cells

We have studied synthesis of specific proteins in two permanent ilness of Friend virus-induced erythroleukemia cells (Friend line 745 and Ostertag line FSD-1, both derived from DBA/2 mice). By 96 hr following treatment with 1–2% dimethyl sulfoxide (Me₂SO), up to 25% of the protein being synthesized by both these cultures is hemoglobin. At that time, hemoglobin constitutes up to 10% of the cellular soluble protein. Both lines synthesize heme and globin coordinately, and α and β globin chains in a nearly balanced 1:1 ratio. However, the ratio of β^Major:β^Minor chains synthesized by these induced Friend leukemia (FL) cells is approximately 9 in the FSD-1 line and 1.3 in the Friend Clone 745 line, whereas it is 4 in normal adult DBA/2 mouse erythrocytes. Evidence for the latter conclusion was obtained by electrophoresis of FL hemoglobins on cellulose acetate membranes, and also by chromatographic separation of α, β^Major, and β^Minor globins on carboxymethylcellulose in 8 M urea at 20°C. Carbonic anhydrase activity per mg protein is 3 times higher in induced than in control cultures. 2,3-diphosphoglyceric acid is not found in induced FL cells. Induced and control FL cells agglutinate strongly and equally with Phaseolus vulgaris phytohemagglutinin. The developmental process in these cultured leukemia cells appears to be an aberrant erythropolesis.     

The roles of RNA polymerase and RNAase I in stable RNA degradation in Escherichia coli carrying the srnB+ gene

In Escherichia coli cells carrying the srnB⁺ gene of the F plasmid, rifampin, added at 42°C, induces the extensive rapid degradation of the usually stable cellular RNA (Ohnishi, Y., (1975) Science 187, 257–258; Ohnishi, Y., Iguma, H., Ono, T., Nagaishi, H. and Clark, A.J. (1977) J. Bacteriol. 132, 784–789). We have studied further the necessity for rifampin and for high temperature in this degradation. Streptolidigin, another inhibitor of RNA polymerase, did not induce the RNA degradation. Moreover, the stable RNA of some strains in which RNA polymerase is temperature-sensitive did not degrade at the restrictive temperature in the absence of rifampin. These data suggest that rifampin has an essential role in the RNA degradation, possibly by the modification of RNA polymerase function. A protein (M_r 12 000) newly synthesized at 42°C in the presence of rifampin appeared to be the product of the srnB⁺ gene that promoted the RNA degradation. In a mutant deficient in RNAase I, the extent of the RNA degradation induced by rifampin was greatly reduced. RNAase activity of cell-free crude extract from the RNA-degraded cells was temperature-dependent. The RNAase was purified as RNAase I in DEAE-cellulose column chromatography and Sephadex G-100 gel filtration. Both in vivo and with purified RNAase I, a shift of the incubation mixture from 42 to 30°C, or the addition of Mg²⁺ ions, stopped the RNA degradation. Thus, an effect on RNA polymerase seems to initiate the expression of the srnB⁺ gene and the activation of RNAase I, which is then responsible for the RNA degradation of E. coli cells carrying the srnB⁺ gene.     

Expression of srnB gene of F plasmid by altered RNA polymerase in Escherichia coli

Degradation of otherwise stable rRNA and tRNA takes place in the presence of rifampin, dependent on the F plasmid srnB gene. We have reported that a protein newly synthesized in the presence of rifampin might be a product of the srnB gene required for stable RNA degradation (Ito, R. and Ohnishi, Y. (1983) Biochim. Biophys. Acta 739, 27–34). Here we have further studied the mechanism of srnB expression. Among eighteen mutants with altered RNA polymerase, two (TJ2470 (rpoC4) and TJ302 (rpoC56)) showed RNA degradation at high temperature (42°C) when the srnB gene was present. Labeling proteins at 42°C in strain TJ2470 indicated that a protein of molecular weight 12 000 was a product of the srnB gene, and that expression of the srnB gene provoked RNA degradation. Using plasmid pTK4, in which the srnB gene is inserted downstream of the promoter of lacZ, lac promoter-dependent expression of the srnB gene, with production of the putative protein product, also induced RNA degradation at 42°C, with no requirement for added rifampin or altered RNA polymerase. RNA degradation in these conditions was quite similar to that in the case of the addition of rifampin; e.g., it showed some responses to Mg²⁺, temperature and RNAase I content of the cells. Expression of the srnB gene dependent on lac promoter was also observed in minicells. Thus, it is inferred that the srnB gene is probably repressed under normal conditions with its own promoter; its expression initiates RNA turnover.     

Replication of RNA viruses: specific binding of the Q RNA polymerase to Q RNA

The specific binding in vitro of the Qβ RNA polymerase to Qβ RNA has been detected by the formation of an enzyme-Qβ RNA complex that did not exchange bound RNA molecules and was not dissociated by 0.8 m NaCl. Formation of this nondissociating complex required GTP and two host protein factors, but not ATP, CTP, UTP, or Mg²⁺ ions. GDP, GMP, dGTP, ITP, and β,γ-methylene GTP did not replace GTP in the reaction. Complex formation at 0 °C was not observed, and the rates of the reaction at 30 °C and 25 °C were 41% and 23%, respectively, of the rate at 37 °C. The reaction occurred with intact Qβ RNA and with polycytidylic acid template but not with bacterial or other bacteriophage RNA. With limiting amounts of enzyme, the amount of Qβ RNA bound in the nondissociating complex was the same as the amount of [γ-³²P]GTP incorporated into nascent RNA chains, indicating a close relationship between complex formation and the initiation of RNA synthesis. The two reactions appear to be separate, however, because in the absence of Mg²⁺ ions, when complex formation occurred readily, no RNA synthesis could be detected either by incorporation of labeled substrate into acid-insoluble material or by formation of short RNA chains still attached to the enzyme. In the presence of factor protein and GTP, a maximum of one active enzyme molecule was bound per molecule of Qβ RNA template, as determined by a liquid polymer phase-separation procedure. These results suggest that formation of the nondissociating complex measures recognition by the Qβ RNA polymerase of a single Qβ RNA site utilized for the initiation of synthesis.     

Characterisation of rinderpest virus RNA and the action of actinomycin D on its replication

RNA extracted from purified rinderpest virus was characterised by sucrose gradient sedimentation and polyacrylamide gel electrophoresis. The predominant virion RNA species had a sedimentation constant of 46S and its estimated molecular weight was 4.8 × 10⁶ daltons. Consistently high amounts of UMP and AMP were detected. The melting-temperature profile of the virion RNA suggested absence of secondary structure. The effect of actionomycin D on the replication of rinderpest virus in Vero cells was studied by following the viral RNA synthesis using labelled uridine as well as by infectivity titration. The viral RNA synthesis was not affected until 12 h following infection and was inhibited thereafter between 18 and 48 h to an extent of 25% at 5 and 10 Μg levels of the drug. A 100 to 1000-fold reduction in the infectivity titres was observed in the presence of the drug. These results suggest that actinomycin D inhibits rinderpest viral RNA replication. Sedimentation analysis of viral RNA extracted from drug-treated cultures showed inhibition of the genome RNA of rinder-pest virus.     

A selective temperature-sensitive defect in viral RNA expression in cells infected with a ts transformation mutant of murine sarcoma virus

We investigated the nature of the defect in the temperature-sensitive mutant of Moloney murine sarcoma virus (Mo-MuSV), termed ts110. This mutant has a temperature-sensitive defect in a function required for maintenance of the transformed state. A nonproducer cell clone, 6m2, infected with ts110 expresses P85 and P58 at 33°C, the transformed temperature, but only P58 is detected at the restrictive temperature of 39°C. Shift-up (33°C → 39°C) and in vitro experiments have established that P85 is not thermolabile for immunoprecipitation. Previous temperature-shift experiments (39°C → 33°C) have shown that P85 synthesis resumes after a 2–3 hr lag period. Temperature shifts (39°C → 33°C) performed in the presence of actinomycin D prevented the synthesis of P85, whereas P58 synthesis did not decline for 5 hr, suggesting that P58 and P85 are translated from different mRNAs. The shift-up experiments also indicated that, once made, the RNA coding for P85 can function at the restrictive temperature for several hours. MuSV-ts110-infected cells superinfected with Mo-MuLV produced a ts110 MuSV-MuLV mixture. Sucrose gradient analysis of virus subunit RNAs revealed a ～28S and a ～35S peak. Electrophoresis of the ～28S poly(A)-containing RNA from ts110 virus in methyl mercuric hydroxide gels resolved two RNAs with estimated sizes of 1.9 × 10⁶ and 1.6 × 10⁶ daltons, both smaller than the wild type MuSV-349 genomic RNA (2.2 × 10⁶ daltons). RNA in the ～28S size class from virus preparations harvested at 33°C was found to translate from P85 and P58, whereas, the ～35S RNA yielded helper virus Pr63^gag. In contrast, virus harvested at 39°C was deficient in P85 coding RNA only. Peptide mapping experiments indicate that P85 contains P23 sequences, a candidate Moloney mouse sarcoma virus src gene product. Taken together, these results suggest that two virus-specific RNAs are present in ts 110-infected 6m2 cells and rescued ts110 pseudotype virions at 33°C, one coding for P85, whose expression can be interfered with by shifting the culture to 39°C; the other coding for P58, whose expression is unaffected by temperature shifts. P85 is a candidate gag-src fusion protein, while P58 contains gag sequences only.     

Production of 10-hydroxy-12,15(Z,Z)-octadecadienoic acid from α-linolenic acid by permeabilized cells of recombinant Escherichia coli expressing the oleate hydratase gene of Stenotrophomonas maltophilia

Recombinant Escherichia coli, expressing the oleate hydratase gene of Stenotrophomonas maltophilia, was permeabilized by sequential treatments with 0.125 M NaCl and 2 mM EDTA. The optimal conditions for the production of 10-hydroxy-12,15(Z,Z)-octadecadienoic acid from α-linolenic acid by permeabilized cells were 35 °C and pH 7.0 with 0.1 % (v/v) Tween 40, 50 g permeabilized cells l^?1, and 17.5 g α-linolenic acid l^?1. Under these conditions, permeabilized cells produced 14.3 g 10-hydroxy-12,15(Z,Z)-octadecadienoic acid l^?1 after 18 h, with a conversion yield of 82 % (g/g) and a volumetric productivity of 0.79 g l^?1 h^?1. These values were 17 and 168 % higher than those obtained by nonpermeabilized cells, respectively. The concentration, yield, and productivity of 10-hydroxy-12,15(Z,Z)-octadecadienoic acid obtained by permeabilized cells are the highest reported thus far.